Abstract

   Cervical cancer is one of the most common malignant tumors among women
   in the world. In clinical practice, Hedyotis diffusa has
   pharmacological effects in treating cervical cancer, but its components
   are relatively complex, and the mechanism of Hedyotis diffusa in
   treating cervical cancer is still unclear. In this work, the potential
   active components and mechanism of Hedyotis diffusa in the treatment of
   cervical cancer were explored by means of network pharmacology. By
   constructing its active ingredient-target network, and enriching and
   analyzing the targets, we found the key targets and their effective
   components (beta-Sitosterol and Quercetin) that play a therapeutic
   role. Finally, we evaluated the prognostic value of the core target
   genes through survival analysis. Our work initially explored the
   therapeutic mechanism of cervical cancer, which lays a theoretical
   foundation for further exploring its pharmacological action and its
   clinical application.

   Keywords: hedyotis diffusa, component, cervical cancer, mechanism,
   network pharmacology

Introduction

   Cervical cancer is the fourth most common female malignant tumor and
   the most common cause of cancer death in the world ([38]Bray et al.,
   2018). About half a million women develop cervical cancer every year
   and about 300,000 people die from the disease every year ([39]Cohen et
   al., 2019). The incidence and mortality rates of cervical cancer in
   developed countries are much lower than those in developing countries,
   which is mainly due to the lack of screening and prevention programs
   for cervical cancer in many developing countries ([40]Denny, 2012).
   Cervical cancer is considered as the only one which is known cause and
   can be preventable among human cancers. Although we know that the
   high-risk subtype of human papillomavirus (HPV) is the main cause of
   cervical cancer, the incidence and mortality of cervical cancer have
   not been significantly reduced ([41]Walboomers et al., 1999). Cervical
   cancer patients are mainly treated by surgery, radiotherapy and
   chemotherapy. And novel immunotherapy, such as antibody targeted
   therapy and adoptive cell therapy, has unsatisfactory efficacy
   ([42]Dyer et al., 2019). Surgical resection of tumors is mainly applied
   to early-stage patients, but most patients diagnosed with cervical
   cancer are generally advanced and lose the best treatment time, so
   chemotherapy and radiotherapy are the main treatment methods ([43]Li et
   al., 2016). However, most chemotherapeutic drugs have severe toxic and
   side effects and long-term use will result in drug resistance, which
   seriously affect the efficacy. By contrast, Chinese medicinal active
   ingredients have the advantages of multiple pathways, multiple targets
   and small adverse reactions in the anti-tumor aspect, and may be more
   suitable for clinical treatment of cancer patients ([44]Nie et al.,
   2016). Hedyotis diffusa is panicum miliaceum of Rubiaceae, widely
   distributed in subtropical regions ([45]Li et al., 2013), and it is a
   famous traditional Chinese medicine for the treatment of
   inflammation-related diseases such as hepatitis and appendicitis in
   China ([46]Chen et al., 2016). Herba hedyotidis diffusae has various
   pharmacological activity, such as anticancer activity ([47]Liu et al.,
   2010), antioxidant activity ([48]Lu et al., 2000),immunomodulatory
   effect ([49]Lin CC. et al., 2011) and anti-inflammatory activity
   ([50]Ye et al., 2015),etc. At present, more and more clinical
   researches prove that the oldenlandia diffusa has remarkable anticancer
   activity ([51]Gupta et al., 2004). Qiao Yan Cai et al. studied the
   molecular mechanism of Hedyotis diffusa in the treatment of colon
   cancer, and the results showed that Hedyotis diffusa exerts the
   curative effect mainly by regulating the expression of several
   important genes in the Signal Transducer and Activator of Transcription
   3 signaling pathway. Hedyotis diffusa can inhibit the phosphorylation
   of Signal Transducer and Activator of Prescription 3, thereby promoting
   the apoptosis of tumor cells and inhibiting the proliferation of tumor
   cells to achieve the curative effect of treating colon cancer ([52]Cai
   et al., 2012). Studies have shown that one of the mechanisms of
   Hedyotis diffusa’s participation in tumor treatment is to inhibit tumor
   angiogenesis ([53]Lin J. et al., 2011). As for the mechanism of
   treating cervical cancer,proto-oncogene c-Sre is a member of
   Sre-familykinases, which is a kind of non-receptor protein tyrosine
   kinase and plays an important role in regulating cell growth,
   development, differentiation and other biological functions. Studies
   have shown that proto-oncogene c-Sre can induce malignant
   transformation of cells. Studies have found that down-regulating the
   activity of c-Sre kinase can effectively inhibit the proliferation of
   cervical cancer cells ([54]Kong et al., 2011). However, due to the
   complex components of Hedyotis diffusa, the mechanism for its treatment
   of most cancers including cervical cancer is not clear. Network
   pharmacology was first proposed by the British pharmacologist Hopkins
   in 2007. It is a new subject based on system biology theory and
   multi-directional pharmacology theory, which integrates information
   network science and computer science. And it has the overall and
   systematic research characteristics ([55]Hopkins, 2008). Network
   pharmacology can be used to analyze biological system networks, predict
   and infer the correlation between targets and networks, and then
   construct a “drug-target-disease” interaction network, which can
   comprehensively and systematically clarify the intervention and
   influence of drugs on diseases ([56]Zhang et al., 2014). In this study,
   with the help of network pharmacology, the effective active compounds
   of Hedyotis diffusa for treating cervical cancer were systematically
   screened out, and the pharmacological effects of Hedyotis diffusa for
   treating cervical cancer with multiple targets, multiple pathways and
   multiple mechanisms were preliminarily revealed.

Methods

Collection of Effective Components and Targets

   In TCMSP ([57]http://tcmspw.com/tcmsp.php) database, the chemical
   components of Herba Hedyotis were collected by retrieving the key word
   “Herba Hedyotis” and taking oral bioavailability greater than or equal
   to 30% and drug-like property of 0.18 as screening conditions, the
   ineffective components were removed. Finally, target information
   corresponding to the active components is collected through HERB
   ([58]http://herb.ac.cn/) database and the network of component targets
   is visualized by Cytoscape.

Acquisition of Cervical Cancer-Related Targets

   By searching the keyword “cervical cancer” in GeneCards database
   ([59]https://www.genecards.org/), we collected 7,110 related targets.
   Finally, we used Venn diagram to display the disease targets and drug
   component action targets, and got 175 common targets.

Functional Enrichment Analysis

   Functional enrichment analysis included the GO functional enrichment
   analysis and KEGG pathway analysis. The GO functional enrichment
   analysis mainly included three parts: biological process (BP),
   molecular function (MF), cellular component (CC). Pathway enrichment
   analysis and GO enrichment analysis were performed using DAVID database
   to obtain the results of the top 20 FDR rankings, respectively, and the
   enrichment results were visualized using BioFormatics
   ([60]www.bioinformatics.com.cn).

Construction of Protein Interaction Network and Extraction of Core Targets

   Import the intersection targets into STRING database and build PPI
   network. Then, using cytoHubba in Cytoscape to extract the top ten
   targets in PPI network by MCC algorithm, and then performing pathway
   enrichment analysis on the extracted core targets.

Prognostic Information of Core Targets

   KaplanMeie-plotter is a popular website tool based on EGA, TCGA and GEO
   databases, which is used to evaluate the survival of gene pairs. The
   prognosis of 10 core targets in cervical cancer was obtained by GEPIA2
   (gepia2. cancer-pku.cn), and the targets with p value less than or
   equal to 0.05 were selected.

Results

Related Targets of Hedyotis Diffusa in the Treatment of Cervical Cancer

   We screened 7,110 cervical cancer-related targets from Gene Cards
   database. Six components of Hedyotis diffusa and its 203 action targets
   were collected through TCMSP database and HERB database, among which
   there were 175 cervical cancer-related targets ([61]Figure 1). Here, we
   show the interaction between components and targets in the form of
   network, as shown in [62]Figure 1. Red squares represent drug
   components, and circles represent component action targets, among which
   orange targets represent cervical cancer-related targets and green
   circles represent non-cervical cancer-related targets. It can be seen
   that most of the targets in the whole target network are related to
   cervical cancer, suggesting that Hedyotis diffusa has certain
   pertinence in the treatment of cervical cancer.

FIGURE 1.

   [63]FIGURE 1
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   Screening of therapeutic targets and construction of its
   component-target network.

Functional Enrichment Analysis of Therapeutic Targets for Cervical Cancer

   GO function enrichment and KEGG pathway enrichment of the above 175
   common targets were analyzed by DAVID database
   ([65]https://david.ncifcrf.gov/home.jsp) ([66]Figure 2). GO analysis
   includes biological processes (BP), cellular components (CC) and
   molecular functions (MF), which together describe the functions of gene
   products.

FIGURE 2.

   [67]FIGURE 2
   [68]Open in a new tab

   KEGG pathway analysis and GO enrichment analysis.

   As shown in [69]Figure 2, the pathway enrichment analysis of Hedyotis
   diffusa in treating cervical cancer mainly involves Pathways in cancer,
   Hepatitis B, Bladder cancer, Prostate cancer, Pancreatic cancer, TNF
   signaling pathway, HIF-1 signaling pathway, Non-small cell lung cancer,
   Chagas disease (American trypanosomiasis), Leishmaniasis and so on. GO
   analysis mainly involves response to drug, positive regulation of
   transcription from RNA polymerase II promoter, positive regulation of
   transcription, DNA-templated, positive regulation of gene expression,
   extracellular space, membrane raft, cell surface, nucleoplasm, enzyme
   binding, protein binding, transcription factor binding, etc.

   Enrichment analysis showed that Hedyotis diffusa may act on multiple
   targets through various signal pathways and play a role in the
   treatment of cervical cancer. At the same time, it also provides
   reference value for further searching for key core targets and
   compounds.

Construction of Protein-Protein Interaction Network and Analysis of its Core
Targets

   We introduced the screened 175 common target genes into STRING, and set
   the minimum confidence level to 0.4. PPI network was constructed by the
   algorithm of STRING database, and the relationship between protein was
   visualized in the form of network. As shown in [70]Figure 3, the
   network graph has 175 nodes and 3,424 edges, and the average node
   degree is 39.1, and the p value is less than 1.0e-16. Nodes represent
   target proteins, and each line represents the interaction between
   target proteins. The thickness of the connecting line represents.

FIGURE 3.

   [71]FIGURE 3
   [72]Open in a new tab

   Protein-protein interaction network.

   In addition, cytoHubba was used for topology analysis of PPI networks,
   and the top ten core targets were extracted, which were successively
   AKT1, VEGFA, TNF, IL6, PTGS2, JUN, IL1B, CASP3, MAPK3, and
   MMP9([73]Figure 4). Later, we further enriched the pathways for the
   core targets, with the following results: TNF signaling pathway,
   Hepatitis B, Pertussis, Pathways in cancer, Chagas disease (American
   trypanosomiasis), etc. We found that the first-ranked pathways not only
   had the most significant statistical significance, but also involved
   more core targets. In addition, this pathway also appeared in our
   previous enrichment results of the entire PPI network, and it was
   ranked high, suggesting that Hedyotis diffusa may play an important
   role by mediating multiple pathways in the treatment of cervical
   cancer, and TNF signaling pathway may play a key role in the entire
   treatment network.

FIGURE 4.

   [74]FIGURE 4
   [75]Open in a new tab

   Core target network and its pathway analysis.

Prognostic Analysis of Core Targets and Construction of Component Target
Network

   In order to further determine the role of the core target in the
   network, we analyzed the overall survival of the core target by using
   GEPIA2. As shown in [76]Figure 5, the results show that four of the 10
   core targets have prognostic value in cervical cancer, namely VEGFA,
   IL1B, IL6, and JUN. The prognosis of all these targets is poor (HR =
   1.7), and the low expression in cervical cancer has a good overall
   survival time. It also suggests that the components interacting with
   these targets may play the role of small molecule inhibitors, thus
   mediating the development of cervical cancer.

FIGURE 5.

   [77]FIGURE 5
   [78]Open in a new tab

   Overall survival of core target.

   In addition, we also constructed the core target-related component
   network ([79]Figure 6). Only four components (C3, C4, C5, C6) were
   found to act on the core targets, among which C5(beta-Sitosterol) and
   C6(Quercetin) acted on the targets with poor prognosis. In addition,
   whether in the previous component-target network ([80]Figure 1), or in
   the core target component network ([81]Figure 6). Compared with other
   components, both C5 and C6 have larger degree value and more targets,
   suggesting that these two components play a leading role in the whole
   network. The above analysis results show that various active
   ingredients contained in Hedyotis diffusa interfere with the progress
   of cervical cancer by acting on its target network to mediate various
   pathways, thus exerting curative effects. Among them, beta-Sitosterol
   and Quercetin may play a key role, and the analysis results need
   further verification by subsequent experiments.

FIGURE 6.

   [82]FIGURE 6
   [83]Open in a new tab

   Core target component network and chemical structures of C5 and C6.

Discussion

   Cervical cancer is a common gynecological malignant tumor, with the
   mortality rate second only to that of ovarian cancer, and it is one of
   the malignant tumors threatening the health of women. Western medicine
   currently mainly adopts radiotherapy, chemotherapy and surgery, while
   radiotherapy and chemotherapy tolerance has become one of the main
   causes for the treatment failure, cancer metastasis and recurrence of
   cervical cancer ([84]Vänskä et al., 2021). In recent years, Chinese
   medicine has been widely used in clinical due to its advantages of
   multi-channel, multi-target and small adverse reactions. Modern studies
   have shown that Hedyotis diffusa has the effects of anti-tumor,
   anti-oxidation, anti-inflammation and enhance the body’s non-specific
   immune function. Hedyotis diffusa is often used to treat various tumors
   in clinic, especially gynecological tumors like cervical cancer, and
   has obtained good clinical efficacy. However its mechanism of action in
   the treatment of cervical cancer has not yet been elucidated. Based on
   the multi-component, multi-target, multi-channel research ideas, this
   work elucidated the potential mechanism of Hedyotis diffusa in the
   treatment of cervical cancer from the microscopic point of view by
   means of network pharmacology. In this study, six important active
   components related to cervical cancer were screened from Hedyotis
   diffusa Willd, which correspond to 175 targets of cervical cancer. The
   six important active ingredients in Hedyotis diffusa for treating
   cervical cancer were beta-Sitosterol,Quercetin,
   Poriferasterol,Stigmasterol, 2-methoxy-3-methyl-9,10-anthraquinone and
   (4aS,6aR,6aS,6bR,8aR,10R,12aR,14bS)-10-hydroxy-2,2,6a,6b,9,9,12a-heptam
   ethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carbo
   xylic acid, of which the first two components played a more important
   role in the treatment of cervical cancer than other components.
   Quercetin is a kind of flavonoid, which widely exists in many fruits
   and vegetables in nature, and has many pharmacological activities such
   as anti-tumor, anti-virus, anti-inflammation, anti-oxidation and so on
   ([85]Gibellini et al., 2011). Madhumitha Kedhari Sundaram et al.
   studied the mechanism of quercetin in the treatment of cervical cancer
   through relevant experiments. The results showed that quercetin could
   induce DNA damage, reduce the activity of cancer cells, and promote the
   apoptosis of cancer cells by promoting G2-M cell cycle arrest. In
   addition, quercetin can also block PI3K and other signaling pathways
   and inhibit the activity of anti-apoptotic proteins to promote cancer
   cell apoptosis ([86]Darband et al., 2020). Related studies have shown
   that beta-Sitosterol has curative effect on a variety of cancers, such
   as cervical cancer, breast cancer, colon cancer, prostate cancer, and
   so on ([87]Choudhary and Tran, 2011). Studies by Dali Cheng et al. have
   shown that beta-Sitosterol can inhibit the proliferation of cervical
   cancer cells and promote the apoptosis of cervical cancer cells. which
   is mainly related to the expression of P53 and HPVE six proteins
   ([88]Cheng et al., 2015). The core targets of Hedyotis diffusa in
   treating cervical cancer mainly included AKT1, VEGFA, TNF, IL6, PTGS2,
   JUN, IL1B, CASP3, MAPK3, and MMP9. In addition, by analyzing the
   survival prognosis of the core targets, we found that the four genes
   with poor prognosis were namely VEGFA, IL1B, IL6 and JUN. VEGFA, as a
   member of VEGF family, is the most potential signaling protein to
   promote angiogenesis ([89]Bagri et al., 2010). Baohuan Chen et al.
   found that VEGFA may inhibit the proliferation and metastasis of
   cervical cancer cells through Akt/mTor/PI3k signaling pathway ([90]Chen
   et al., 2014). IL6, a multifunctional cytokine, plays an important role
   in regulating the growth of tumor cells ([91]Kishimoto, 1989). Lin-Hung
   Wei et al. showed that IL-6 can regulate the expression of apoptosis
   protein Mcl-1 in cervical cancer cells and promote the occurrence of
   cervical cancer ([92]Wei et al., 2001). Recent studies have shown that
   mi R-125 can inhibit the progression of cervical cancer by inhibiting
   the VEGF and PI3K-Akt signaling pathways ([93]Fu et al., 2020). IL1B, a
   pro-inflammatory cytokine, is related to tumorigenesis. Studies have
   shown that the level of IL1B in plasma of patients with cervical cancer
   was significantly higher than that of the control group, and the
   expression level of IL1B played an important role in the carcinogenesis
   of cervical cancer ([94]Qian et al., 2010). In order to further verify
   our screening results, we selected C6- quercetin, an important active
   compound in the treatment of cervical cancer by Hedyotis diffusa, for
   molecular docking with the core targets IL1B and IL6 which have
   prognostic value for cervical cancer respectively. Generally, when the
   binding energy is less than 0, the compound and protein can
   spontaneously combine, and the lower the binding energy, the greater
   the possibility of interaction between the compound and protein.
   Docking score ≤ −5.0 kJmol−1 indicates that it has good binding
   activity. The molecular docking results are shown in [95]Figure 7,
   IL1B-quercetin (−8.2) and IL6-quercetin (−7.1). The results show that
   the core component C6- quercetin predicted in this study has strong
   binding ability with key targets (IL1B and IL6) and the binding ability
   of C6- quercetin with target IL1B is stronger, which further confirms
   the reliability of the prediction results of network pharmacology. The
   main signal pathways involved in the treatment of cervical cancer by
   Hedyotis diffusa were pathways in cancer, hepatitis B and blader C.
   Pathways in cancer, Hepatitis B, Bladder cancer, Prostate cancer,
   Pancreatic cancer, TNF signaling pathway, HIF-1 signaling pathway,
   Non-small cell lung cancer, Chagas disease (American trypanosomiasis),
   Leishmaniasis and so on.

FIGURE 7.

   [96]FIGURE 7
   [97]Open in a new tab

   Molecular docking of IL1B-Quercetin (A) and IL6_Quercetin (B).

   Through the above analysis, we finally screened the active product
   capable of interfering with the progression of cervical cancer from
   Herba Hedyotis, and initially explored the multi-pathway action
   mechanism of Herba Hedyotis in the treatment of cervical cancer by the
   analysis method of network pharmacology, which provided reference value
   and theoretical basis for future clinical research. The experimental
   research method is reasonable, but the potential mechanism of Melastoma
   dodecandrum against cervical cancer is not yet clear, and further
   experiments are needed.

Data Availability Statement

   The original contributions presented in the study are included in the
   article/Supplementary Material, further inquiries can be directed to
   the corresponding authors.

Author Contributions

   All authors listed have made a substantial, direct, and intellectual
   contribution to the work and approved it for publication.

Funding

   This study was supported by Key projects of workstation of He Lin
   (Grant No. 18331101) Natural Foundation of Zhejiang Provincial China
   (LGF18H300005) and Natural Foundation of Jiangxi Provincial China
   (20202BABL216078\20195656).

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Publisher’s Note

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References